The biological function of nucleic acid binding proteins is often separable from their sequence specificity. In some cases, as with many RNA and DNA polymerases, a protein possessing the enzymatic activity site is directed to the correct location through its interaction with a distinct polypeptide. In others, as with many transcription factors and regulators of mRNA metabolism, function and sequence specificity reside in distinct domains of the same polypeptide. Dissection of the two domains from one another facilitates their detailed analysis, liberating each from the constraints of the other.
Poly(A) is a virtually universal feature of mRNAs in eukaryotes, and exists in the cytoplasm as an RNA-protein complex with Poly(A) binding protein (Pab1p). Like the tail, Pab1p is highly conserved. The presence of a poly(A) tail typically stabilizes mRNAs and can enhance their translation both in vivo and in vitro. It is unclear whether these biological functions reside solely in the protein, or require the RNA-protein complex.
The role of the Pab1p in stability has been demonstrated in the budding yeast Saccharomyces cerevesiae. The yeast PAB1 gene is required for viability (Sachs, 1986). The lethality of a pab1 deletion can be suppressed by mutations within various genes associated with mRNA turnover, such as xrn1 and dcpl, indicating Pab1p likely has an essential function in mRNA decay (Hatfield, 1996). Degradation of most yeast mRNAs is initiated by shortening of the 3' poly(A) tail, followed by Dcplp cleavage of the 5'm.sup.7 GpppG cap ("decapping"), which permits 5' to 3' exonucleolytic digestion by the XRN1 gene product (Decker, 1993; Muhlrad, 1994; Beelman, 1996). Decapping occurs only after the poly(A) tail has been shortened to 10-12 adenosine residues (Muhlrad, 1994), which corresponds provocatively with the minimal Pab1p binding site of 12 adenosines and suggests that eviction of the last Pab1p/poly(A) complex triggers turnover. Furthermore, in the absence of Pab1p, decapping becomes independent of poly(A) length (Caponigro, 1995).
The molecular link between poly(A) and the cap may be a tripartite protein bridge between the two ends of the mRNA. Pab1p, when bound to the poly(A) tail, interacts in vitro with two yeast proteins, TIF4631 and TIF4632, related to the translation initiation factor eIF-4G (Tarun, 1996). eIF-4G in turn interacts with eIF-4F, containing the cytoplasmic cap-binding protein, eIF-4E, an initiation factor itself. The in vitro association of Pab1p and eIF-4G is dependent on the poly(A) tail, and is consistent with earlier work demonstrating biochemical interactions between poly(A) and complexes containing eIF-4F in vitro (Gallie, 1994). The same tripartite bridge could explain poly(A) 's stimulation of translation in vitro and in vivo.
The interaction between Pab1p and eIF-4G, demonstrated in vitro, requires the presence of RNA, implying that the poly(A)/Pab1p complex likely is essential. Similarly, deadenylation in crude yeast extracts is dependent on the presence of Pab1p, implying that the yeast deadenylase may recognize not poly(A) alone, but the poly(A)/Pab1p complex (Lowell, 1992).